1. Field of the Invention
The field of this invention is techniques to modulate the dynamics of transcriptionally regulated protein secretion and mRNA stability. These techniques are of particular use for improving the dynamics of insulin secretion, which is a useful tool in cell-based therapies for insulin-dependent diabetes.
2. Background Art
Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Cell sourcing constitutes a critical issue in developing a cell-based therapy for treatment of IDD. Genetic engineering of non-β cells, particularly those of hepatic origin, for glucose-responsive insulin secretion offers significant promise in developing a cell-based therapy for insulin-dependent diabetes. Responsiveness to physiologic stimuli is introduced at the gene transcription level by using promoters up-regulated by glucose and possibly down-regulated by insulin (Thulé, et al., 2000 Gene Ther. 7:205-14; Thulé, P. M. & Liu, J. M., 2000 Gene Ther. 7:1744-52; Lee, et al., 2000 Nature 408:483-8; Barry, et al., 2001 Hum. Gene Ther. 12:131-9; Chen, et al., 2001 Mol. Ther. 3:584-90).
Based on this concept, glucose-regulated insulin expression has been achieved in streptozotocin (STZ)-induced diabetic rodents (Thulé, P. M. & Liu, J. M., 2000 Gene Ther. 7:1744-52; Lee, et al., 2000 Nature 408:483-8; Barry, et al., 2001 Hum. Gene Ther. 12:131-9; Chen, et al., 2001 Mol. Ther. 3:584-90). A major advantage of these cells is that they are potentially autologous, retrieved as a biopsy from the patient. A disadvantage of these cells is that transcriptionally controlled cells exhibit sluggish secretion dynamics and thus may not be suitable, as such, for achieving normoglycemia in higher diabetic animals and humans. To expedite the dynamics of secretion down-regulation, translation needs to stop soon after transcription has been turned off. Of particular significance is the slow dynamics of secretion down-regulation, which result in the cells secreting insulin long after the stimulus has been removed and treatment with these cells may thus revert diabetes to hyperinsulinemia and hypoglycemia, a serious pathological condition.
It has been suggested that the prolonged stability of preproinsulin (PPI) mRNA causes the sluggishness of secretion down-regulation (Efrat, S., 1998, Diabetologia 41:1401-9; Dong, H. & Woo, S. L., 2001 Trends Endocrinol. Metab. 12:441-6). Although prior reports on the PPI MRNA half-life in non-β cells are limited, data from normal and transformed β cells strongly indicate that the stability of PPI mRNA is a limiting factor in expediting secretion down-regulation. In isolated primary rat islets, the half-life of PPI mRNA was estimated to be 77 hours under high glucose (17 mM) and 29 hours under low glucose concentration (3.3 mM) (Nielsen, et al., 1985, J. Biol. Chem. 260:13585-9). In βTC-3 insulinomas, there was only marginal PPI mRNA degradation over 24 hours after transcription was stopped (Schuppin, G. T. & Rhodes, C. J., 1996, Biochem. J. 313:259-68), while the half-life of PPI mRNA in RIN-5F insulinomas was found to be 58 hours and 26 hours under high (20 mM) and low (3 mM) glucose concentration, respectively (Nielsen, et al., 1985, J. Biol. Chem. 260:13585-9).
The topic of modulation of mRNA stability is currently under intense investigation (Guhaniyogi, J. & Brewer, G., 2001, Gene 265:11-23; Clayton, C.E., 2002, Embo. J. 21:1881-8; van Hoof, A. & Parker, R., 2002, Curr. Biol. 12:R285-7). Specifically with PPI mRNA, to accelerate the rate of mRNA turnover, the use of anti-sense RNA (Taniguchi, K., et al., 1996, Cell Transplant 5:S55-7) or connecting the insulin gene with the 3′-untranslated region (3′-UTR) of some labile mRNAs, such as those encoding cytokines, have been considered (Dong, H. & Woo, S. L., 2001, Trends Endocrinol. Metab. 12:441-6). However, these systems require the use of more than one construct, or have not shown to be entirely successful.
Recently, nonsense-mediated mRNA decay (NMD) has received significant attention because of its biological and medical importance (Hentze, M. W. & Kulozik, A. E., 1999, Cell 96:307-10; Wilusz, C. J., Wang, W. and Peltz, S. W., 2001, Genes Dev. 15:2781-5; Byers, P. H., 2002, J. Clin. Invest. 109; 3-6; Maquat, L. E., 2002, Curr. Biol. 12, R196-7). Mutant mRNAs with premature stop codons can be detected by cells via a surveillance mechanism, and are subjected to NMD (Li, S. & Wilkinson, M. F., 1998, Immunity 8,135-41; Pulak, R. & Anderson, P., 1993, Genes Dev. 7, 1885-97). NMD likely evolved in vivo to eliminate erratic mRNAs.
Because of the difficulty in modulating the dynamics of protein secretion in general, and insulin secretion specifically, there is a need in the art for techniques to improve the efficiency and dynamics of protein secretion from a recombinant cell.